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Mobilization with granulocyte colony-stimulating factor blocks medullar erythropoiesis by depleting F4/80+VCAM1+CD169+ER-HR3+Ly6G+ erythroid island macrophages in the mouse

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Similar to other tissues, the bone marrow (BM) contains subsets of resident tissue macrophages which are essential to maintain bone formation, functional hematopoietic stem cell (HSC) niches, and erythropoiesis. Pharmacological doses of granulocyte colony-stimulating factor (G-CSF) mobilize HSC in part by interfering with HSC niche-supportive function of BM resident macrophages. As BM macrophages are key to both maintenance of HSC within their niche and erythropoiesis, we investigated the effect of mobilizing doses of G-CSF on erythropoiesis in mice. We now report that G-CSF blocks medullar erythropoiesis by depleting erythroid island macrophages we identified as co-expressing F4/80, vascular cell adhesion molecule (VCAM)-1, CD169, Ly-6G and the ER-HR3 erythroid island macrophage antigen. Broad macrophage depletion by injecting clodronate-loaded liposomes, or selective depletion of CD169(+) macrophages, also concomitantly depleted F4/80(+)VCAM-1(+)CD169(+)ER-HR3(+)Ly-6G(+) erythroid island macrophages and blocked erythropoiesis. This more precise phenotypic definition of erythroid island macrophages will enable studies on their biology and function in normal setting and diseases associated with anemia. Finally this study further illustrates that macrophages are potent relay of innate immunity and inflammation on bone, hematopoietic and erythropoietic maintenance and agents that affect these macrophages, such as G-CSF, are likely to affect these three processes concomitantly.
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Mobilization with granulocyte colony-stimulating factor blocks medullar
erythropoiesis by depleting F4/80
þ
VCAM1
þ
CD169
þ
ER-HR3
þ
Ly6G
þ
erythroid island macrophages in the mouse
Rebecca N. Jacobsen
a,d
, Catherine E. Forristal
a
, Liza J. Raggatt
b
, Bianca Nowlan
a
, Valerie Barbier
c
,
Simranpreet Kaur
b
, Nico van Rooijen
e
, Ingrid G. Winkler
c
, Allison R. Pettit
b
, and
Jean-Pierre Levesque
a,d
a
Stem Cell Biology Group, Mater Research Institute, University of Queensland, Woolloongabba, Queensland, Australia;
b
Bones and Immunology
Group, Mater Research Institute, University of Queensland, Woolloongabba, Queensland, Australia;
c
Stem Cells and Cancer Group, Mater Research
Institute, University of Queensland, Woolloongabba, Queensland, Australia;
d
School of Medicine, University of Queensland, Saint Lucia, Queensland,
Australia;
e
Department of Molecular Cell Biology, Vrije Universiteit, Amsterdam, The Netherlands
(Received 20 December 2013; revised 25 March 2014; accepted 31 March 2014)
Similarly to other tissues, the bone marrow contains subsets of resident tissue macrophages,
which are essential to maintain bone formation, functional hematopoietic stem cell (HSC)
niches, and erythropoiesis. Pharmacologic doses of granulocyte colony-stimulating factor
(G-CSF) mobilize HSC in part by interfering with the HSC niche-supportive function of BM
resident macrophages. Because bone marrow macrophages are key to both maintenance of
HSC within their niche and erythropoiesis, we investigated the effect of mobilizing doses of
G-CSF on erythropoiesis in mice. We now report that G-CSF blocks medullar erythropoiesis
by depleting the erythroid island macrophages we identified as co-expressing F4/80, vascular
cell adhesion molecule-1, CD169, Ly-6G, and the ER-HR3 erythroid island macrophage anti-
gen. Both broad macrophage depletion, achieved by injecting clodronate-loaded liposomes,
and selective depletion of CD169
+
macrophages, also concomitantly depleted F4/80
+
VCAM-
1
+
CD169
+
ER-HR3
+
Ly-6G
+
erythroid island macrophages and blocked erythropoiesis. This
more precise phenotypic definition of erythroid island macrophages will enable studies on their
biology and function in normal settings and on diseases associated with anemia. Finally, this
study further illustrates that macrophages are a potent relayof innate immunity and inflamma-
tion on bone, hematopoietic, and erythropoietic maintenance. Agents that affect these macro-
phages, such as G-CSF, are likely to affect these three processes concomitantly. Ó2014
ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.
Erythropoiesis, the processes leading to the formation of
blood erythrocytes, is a highly active, life-sustaining pro-
cesses. Blood erythrocytes have a life span of 100–
120 days. As such, a typical adult must produce 2.5 million
erythrocytes per second to replace them. Erythropoiesis in-
volves the differentiation of hematopoietic stem cells
(HSCs) into myeloid-erythroid progenitors in the bone
marrow (BM). These progenitors further commit to the
erythroid lineage throughout the generation of proerythro-
blasts and erythroblasts. After synthesizing large quantities
of hemoglobin, the final stage of erythroblast maturation in-
volves extrusion of the nucleus to form anuclear reticulo-
cytes, which then change shape in the blood to become
fully matured erythrocytes. Microscopy sections of BM
have revealed that maturing erythroblasts rosette around a
central macrophage to form erythroid islands [1–5]. It has
been proposed that these erythroid island macrophages
contribute to the efficient transport of iron to erythroblasts
[2,6], enabling the synthesis of large quantities of hemoglo-
bin as O
2
and CO
2
transport, which depends on Fe
2þ
com-
plexed in the heme molecule. It has been proposed that
these ‘‘nursing’’ macrophages at the center of erythropoietic
islands secrete cytokines essential to erythroblast survival
and maintenance, such as insulin-like growth factor 1
Offprint requests to: Dr. Jean-Pierre Levesque, Mater Research Institute –
The University of Queensland, Translational Research Institute, 37 Kent
Street, Woolloongabba 4102, Australia; E-mail: jplevesque@mmri.mater.
org.au;jp_levesque@yahoo.com
Supplementary data related to this article can be found online at http://
dx.doi.org/10.1016/j.exphem.2014.03.009.
0301-472X/$ - see front matter. Copyright Ó2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc.
http://dx.doi.org/10.1016/j.exphem.2014.03.009
Experimental Hematology 2014;42:547–561
[7,8]. Finally erythroid island macrophages phagocytose
and degrade erythroblast nuclei during nuclear extrusion,
a step necessary to generate anucleated reticulocytes
[9,10]. Although these macrophages are essential for eryth-
ropoiesis, little work has been done on their phenotypic
identification so that they can be studied further. This is
particularly important in disease states where erythropoiesis
is perturbed, such as myelodysplastic syndrome, chronic
and acute myeloid leukemia, and chronic inflammation.
Systemic administration of granulocyte colony-
stimulating factor (G-CSF) mobilizes HSC from the BM
into the blood to harvest large quantities of HSC for subse-
quent transplantation in humans. Granulocyte colony-
stimulating factor causes HSC mobilization by perturbing
HSC niches in the BM [11,12]. This results in the downregu-
lation of cell adhesion molecules such as vascular cell adhe-
sion molecule-1 (VCAM-1) and the chemokine CXC-motif
ligand-12 (CXCL12), which are both essential to HSC reten-
tion within the BM [13–16]. Furthermore, it has recently
been demonstrated that this effect of G-CSF on HSC niches
is mediated in part by a subset of BM macrophages [15–17].
In the mouse HSC niche, supportive macrophages express
the macrophage-specific antigen F4/80 together with the
Ly-6G granulocyte antigen [16,18]. A separate report sug-
gests that HSC niche-supportive macrophages express
CD169, also called sialic acid binding immunoglobulin-
like lectin 1 (Siglec-1) or sialoadhesin [15]. However,
whether F4/80
þ
Ly-6G
þ
macrophages express CD169 has
never been reported. Treatment with G-CSF depletes F4/
80
þ
Ly-6G
þ
macrophages in the BM [16,18], resulting in a
concomitant loss of osteoblasts and bone formation,
decreased expression of VCAM-1 and CXCL12, and HSC
mobilization [15–17]. In support of this notion, three inde-
pendent models of macrophage depletion result in HSC
mobilization in the mouse: (1) in macrophage Fas-induced
apoptosis transgenic mice, (2) by injection of clodronate-
loaded liposomes into wild-type mice, or (3) following selec-
tive depletion of CD169
þ
macrophages in Siglec1
DTR/þ
mice
[15,16]. Considering that macrophages are important regula-
tors of both HSC niches and erythropoiesis, we explored the
effect of mobilizing doses of G-CSF on erythropoiesis and
erythroid island macrophages.
Materials and methods
Mice and treatments
All procedures were approved by the Animal Experimentation
Ethics Committee of the University of Queensland.
We purchased C57BL/6 mice from the Animal Resource
Centre (Perth, Australia). Siglec1
DTR/DTR
mice were obtained
from the Riken Bio Resource Centre (Yokohama, Kanagawa,
Japan) and were bred with wild-type C57BL/6 females to generate
Siglec1
DTR/þ
mice for experimentation. Insertion of the simian
diphtheria toxin receptor (DTR) into the Siglec1 gene was de-
tected by genomic polymerase chain reaction (PCR) on ear clips
using Siglec1 forward primer 50-CAATTTCCGGTGCTTACGGT
G-30,Siglec1 reverse primer 50-CATAGTCTAGGCTTCTGT
GC-30, and DTR primer 50-CCGGAGCTCCTTCACATATTT
GC-30, with an annealing temperature of 60C, giving 530bp
and 788bp bands for Siglec1 wild-type and Siglec1
DTR
alleles,
respectively [19].
All experiments were performed on 8- to 10-week-old male
mice. Mice were injected twice daily subcutaneously, either with
125 mg/kg per injection recombinant human G-CSF (Neupogen,
Thousand Oaks, CA) diluted in saline for injection or with saline,
for up to 4 consecutive days.
For macrophage depletion, dichloromethylene bisphosphonate
(clodronate) (Roche Diagnostics, Mannheim, Germany) was
packaged into liposomes as previously described [20]. Empty lipo-
somes were prepared in the same conditions in phosphate-buffered
saline (PBS) without clodronate. Phagocytic macrophages were
depleted in vivo by retro-orbitally injecting 200 mL/20g body
weight clodronate-loaded liposome suspension every second day.
Control mice were injected with an equivalent volume of saline
or PBS-loaded liposomes.
For selective CD169
þ
macrophage depletion, Siglec1
DTR/þ
mice
and control C57BL/6 wild-type mice were injected intraperitoneally
daily with 10 mg/kg purified diphtheria toxin (DPT) (MBL Interna-
tional, Woburn, MA, USA) diluted in saline for injection.
Tissue sampling
Mice were anesthetized and 1 mL blood collected into heparinized
tubes by cardiac exsanguinations. Spleens were harvested, weighed,
and dissociated twice in 3 mL Iscove modified Dulbecco’s medium
(IMDM) with 10% fetal calf serum (FCS) using a GentleMACS
Dissociator tissue homogenizer with matching C tubes (Miltenyi
Biotec, Cologne, Germany) on ‘‘spleen 3’ setting. Femurs were
cleaned of any remaining muscles with a scalpel and paper towel,
BM cells were flushed out using a 21-gauge needle and a 1 mL sy-
ringe containing 1 mL PBS with 2% FCS. Bone marrow leukocytes
were dissociated by successive pipetting with the mounted syringe.
Flow cytometry
Bone marrow cells and splenocytes were pelleted at 370 gfor 5 mi-
nutes at 4C and resuspended in CD16/CD32 hybridoma 2.4G2
supernatant. For macrophage staining, cells were stained with
CD11b-Billiant Violet 605, anti-F4/80-AlexaFluor647 (clone
CI:A3-1), anti-Ly-6G-PECY7 (clone 1A8), anti-VCAM-1-Pacific
Blue (clone 429), CD169-FITC (clone 3D6.112), biotinylated
ER-HR3 antibody, and streptavidin-PE.
For red cell staining, cells were stained with Ter119-FITC,
CD71-PE, and 50 mM Hoescht33342.
For erythroid island macrophage staining, femurs were split in
halves lengthwise by poking nicks in the bone surface with a
scalpel blade. The exposed BM was then gently removed from
the opened bone with IMDM supplemented with 10% FCS and
a 1-mL-tip pipette to minimize disruption of cell aggregates.
Cell suspension was then gently pipetted approximately 10 times
and resuspended in CD16/CD32 hybridoma 2.4G2 supernatant
containing CD11b-Billiant Violet 605, anti-F4/80-AlexaFluor647
(clone CI:A3-1), anti-Ly-6G-APCCY7 (clone 1A8), anti-VCAM-
1-Pacific Blue (clone 429), CD169-FITC (clone 3D6.112),
Ter119-PECy7, biotinylated ER-HR3 antibody, and streptavidin-
PE. Cell aggregates were gated as the tail of the forward scatter
pulse width to detect erythroid islands [21].
548 R.N. Jacobsen et al./ Experimental Hematology 2014;42:547–561
In all stains, 5 mg/mL 7-amino actinomycin D was added 15 mi-
nutes before acquisition to exclude dead leukocytes. Data were ac-
quired on a CyAn (Dako Cytomation, Glostrup, Denmark) flow
cytometer and analyzed following compensation with single color
controls using FloJo software (Tree Star, Ashland, OR). For sort-
ing, cells were sorted on a FACS Aria cell sorter (BD Biosciences,
San Jose, CA, USA).
All antibodies were purchased from Biolegend (San Diego,
CA, USA) except ER-HR3 and CD169 antibodies, which were
purchased from AbD Serotec (Kidlington, UK).
Immunohistochemistry
Spleens and hind limb bones were fixed in PBS with 4% parafor-
maldehyde at 4C for 4 hours and 24 hours, respectively.
Following bone decalcification, tissues were embedded in paraffin
and 4–5 mm sections were cut and placed on SuperFrost Plus
slides (Menzel, Braunschweig, Germany). Sections were deparaf-
finized and rehydrated with xylene and graded ethanol, then
washed in Tris-buffered saline. After antigen retrieval with
trypsin, a section from each sample was stained with unconjugated
rat anti-ER-HR3. This was then detected using a two-step proce-
dure as previously described [22]. Consecutive sections from
each sample were stained with biotinylated rat anti-Ter119
without antigen retrieval. Primary antibody was detected with
horseradish peroxidase conjugated streptavidin (Dako, Glostrup,
Denmark) and diaminobenzidine (DAB) chromogen. Sections
were counterstained with Hematoxylin.
Double staining was undertaken, with the first stage replicating
the procedure for anti-ER-HR3 single staining. Before DAB devel-
opment, sections were incubated with anti-Ter119-biotinylated
antibody, which was subsequently detected using a Vectorstain
ABC-alkaline phosphatase kit (Vector Laboratories, Burlingame,
CA). We detected ER-HR3 expression by DAB chromogen devel-
opment and Ter119 expression by liquid permanent red (Dako).
Double stained sections were counterstained with methyl green.
Specificity of staining was confirmed by comparison to serial
sections stained with isotype-matched control antibody. All sec-
tions were examined using an Olympus BX-51 microscope with
a DP-70 digital camera and DP controller imaging software
(Olympus, Tokyo, Japan).
Ribonucleic acid extraction and quantitative reverse
transcription polymerase chain reaction
Bone marrow and spleen leukocytes were sorted directly into 2 mL
of Trizol (Life Technologies, Carlsbad, CA, USA). Sort was paused
every 10
5
cells sorted to mix collection tubes containing Trizol
phase with sorted cells in aqueous phase. Collection tubes were
then stored at 80C before extraction of ribonucleic acid
(RNA), following manufacturer’s instructions. Reverse transcription
was performed using iScript cDNA kit (Biorad, Hercules, CA,
USA) per manufacturer’s instructions. Quantitative reverse tran-
scription polymerase chain reaction (qRT-PCR was performed us-
ing Taqman Universal PCR master mix (ABI, Life Technologies,
Carlsbad, CA, USA) for Csf3r gene expression and SYBR Green
PCR Master Mix (ABI) for mouse b2-microglobulin. The protocol
for the PCR consisted of one cycle of 50C (2 min), followed by
95C (10 min), followed by 50 cycles each of 95C (10 sec) and
60C (60 sec). Results were normalized relative to b2-
microglobulin mRNA. Primers were Csf3r Taqman Gene Expres-
sion Assay (Mm00432735_m1) (ABI) and for b2-mciroglobulin:
Forward 50–CTGGTCTTTCTGGTGCTTGTC-30; reverse 50–GT
ATGTTCGGCTTCCCATTC-30.
Statistical analyses
All data are presented as average 6SD. Statistical differences
were calculated with a Student’s ttest.
Results
Granulocyte colony-stimulating factor treatment causes
blood marrow anemia
The first indication that G-CSF treatment was interfering with
erythropoiesis was a marked whitening of the BM flushed
from C57BL/6 mice mobilized for 2 to 6 days with G-CSF
compared with BM flushed from saline-treated control mice
(Fig. 1A). To further examine this, we stained BM cells with
7-amino actinomycin D to exclude dead leukocytes,
Hoechst33342 (Ho) to distinguish nucleated leukocytes and
erythroblasts from enucleated reticulocytes and erythrocytes,
and antibodies specific for the erythroid lineage marker
Ter119 and transferrin receptor CD71 to separate maturational
stages of erythroid differentiation [23,24].
A 4-day G-CSF treatment, which mobilizes HSC into the
blood, reduced the number of Ter119
þ
CD71
þ
erythroblasts
fifteenfold, Ter119
þ
CD71
low
polychromatic erythroblasts
one-point-sevenfold, Ter119
þ
CD71
Ho
þ
orthochromatic
erythroblasts one-point-fourfold, and Ter119
þ
Ho
reticulo-
cytes four-point-fivefold (Fig. 1B and C). In contrast to this
marked reduction in erythroblasts and reticulocytes, the num-
ber of Ter119
low
CD71
þ
Ho
þ
proerythroblasts was increased
four-point-fourfold. To confirm these results, we also stained
BM with CD45, Ter119, and CD44 antibodies. Proerythro-
blasts were gated as CD45
Ter119
low
CD44
bright
.The
different differentiation stages of erythroblasts, reticulocytes,
and erythrocytes were gated within the CD45
Ter 11 9
þ
pop-
ulation according to decreasing CD44 expression and forward
scatter [25,26]. This staining and gating strategy confirmed the
marked reduction in all differentiation stages of erythroblasts,
reticulocytes, and erythrocytes with accumulation of proery-
throblasts (Supplementary Figure E1, online only, available
at www.exphem.org). As a result of decreased medullary
erythropoiesis, hemoglobin concentration in the blood slightly
and significantly decreased from 135.3 63.1 g/L to
116.7 63.1 g/L at day 2 of G-CSF treatment, and it normal-
ized at day 4 of treatment (Fig. 1D). Overall, this suggests
that G-CSF causes a transient blockage in the maturation of
proerythroblasts to erythroblasts, with loss of erythroblasts
and reticulocytes in the BM (Fig. 1BandC).
Loss of F4/80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
macrophages in granulocyte colony-stimulating factor
mobilized bone marrow
We have previously reported that G-CSF treatment causes a
loss of F4/80
þ
Ly-6G
þ
macrophages and collapse of HSC
niche function in the BM [16,18]. Another group recently
reported that selective depletion of CD169
þ
macrophages
549R.N. Jacobsen et al. / Experimental Hematology 2014;42:547–561
had similar effects on HSC mobilization and disruption of
HSC niche function [15]. To determine whether F4/
80
þ
Ly-6G
þ
macrophages or CD169
þ
macrophages include
erythroid island macrophages, we performed additional
phenotyping of these BM leukocytes with the ER-HR3 anti-
body, which identifies macrophages in erythropoietic
islands in the mouse BM in steady-state [5,27,28].We
also assessed VCAM-1, since the interaction between
VCAM-1, expressed by macrophages, and its receptor
integrin a4b1, expressed by erythroblasts, is essential to
erythropoietic recovery following cytotoxic challenge
[29,30].
By flow cytometry, the majority of CD11b
þ
F4/
80
þ
VCAM-1
þ
BM macrophages stained positive for both
CD169 and ER-HR3 antigens (Fig. 2A). These
CD11b
þ
F4/80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
macrophages
could be further subdivided into Ly-6G
and Ly-6G
þ
sub-
sets. Interestingly, a 4-day treatment with G-CSF caused a
Figure 1. G-CSF blocks medullary erythropoiesis. (A) Photograph of mouse femoral BM flushed into 1 mL PBS after 4-day treatment with saline, G-CSF, or
clodronate loaded liposomes. Note the loss of red coloration in mice treated with G-CSF or clodronate-loaded liposomes. (B) Representative flow cytometry
dot-plots of mouse BM after a 4-day treatment with G-CSF or saline. The left dot-plots show Ter119 vs. CD71 amongst Ho
þ
nucleated cells. Proerythroblasts
were gated as Ter119
low
CD71
þ
(population I), erythroblasts as Ter119
þ
CD71
þ
(population II), polychromatic erythroblasts as Ter119
þ
CD71
low
(population
III), and orthochromatic erythroblasts as Ter119
þ
CD71
low
(population IV). In the right dot-plots, reticulocytes are gated as Ter119
þ
Ho
Ter119
þ
CD71
low
(population V). (C) Quantification of erythroid populations in the femoral BM after a 4-day G-CSF or saline treatment. (D) Hemoglobin concentration in the
blood of mice treated with saline, G-CSF for 2 days (G2), or G-CSF for 4 days (G4). Data are mean 6SD of 4 mice per group from one representative
experiment among six independent repeats. Differences were evaluated with a ttest. *p#0.05; **p#0.01; ***p#0.001. G5Treated with G-CSF
for 4 days; G2 5treated with G-CSF for 2 days; G4 5treated with G-CSF for 4 days; Sal 5treated with saline for 4 days.
550 R.N. Jacobsen et al./ Experimental Hematology 2014;42:547–561
50% reduction in F4/80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
macrophages. Within this population, F4/80
þ
VCAM-
1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
macrophages were decreased
thirty-fivefold, leaving very rare macrophages of the pheno-
type in the BM of G-CSF-treated mice, whereas the
number of F4/80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
macrophages remained unchanged (Fig. 2B). Notably,
CD11b
þ
F4/80
Ly-6G
þ
granulocytes were negative for
VCAM-1, CD169, and ER-HR3 antigens. Considering
that CD169 [21], VCAM-1 [30], and ER-HR3 [5,28] anti-
gens are expressed by subsets of macrophages that are func-
tionally important for erythropoiesis, our results provide a
finer mapping of BM macrophage subsets and suggest
that CD11b
þ
F4/80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
macrophages represent erythroid island macrophages. Sec-
ondly, mobilizing doses of G-CSF caused a targeted and
efficient depletion of these erythroid island macrophages,
resulting in defective erythropoiesis.
Figure 2. G-CSF treatment reduces the number of F4/80
þ
VCAM1
þ
CD169
þ
ER-HR3
þ
Ly6-G
þ
erythroid island macrophages in the BM. (A) Representative
flow cytometry dot-plots of mouse BM after a 4-day treatment with G-CSF or saline. BM F4/80
þ
macrophages were gated from CD11b
þ
myeloid cells and
further subgated from expression of VCAM-1, CD169, ER-HR3, and Ly6-G antigens. (B) Quantification of macrophage populations in femoral BM after a 4-
day G-CSF or saline treatment. Data are mean 6SD of 4 mice per group from one representative experiment among four independent repeats. Differences
were evaluated with a ttest. (C) Representative phenotype of BM cell aggregates (with high forward scatter peak width) containing Ter119
þ
erythroid cells
and CD11b
þ
myeloid cells in mice treated for 4 days with saline or with G-CSF. Note that the myeloid cells contained in these aggregates are much enriched
in F4/80
þ
VCAM1
þ
CD169
þ
ER-HR3
þ
Ly6-G
þ
macrophages compared with whole BM in panel A (two independent experiments, n54 per experiment).
551R.N. Jacobsen et al. / Experimental Hematology 2014;42:547–561
To further document the phenotype of erythroid island
macrophages, we harvested BM cells in IMDM containing
Ca
2þ
and Mg
2þ
to preserve cell-cell adhesive interactions
between macrophages and erythroid cells. By gating on
events with larger forward scatter peak width (FSC-W),
we could identify cell aggregates that were positive for
both the erythroid marker Ter119 and myeloid marker
CD11b, as previously reported [21]. These
Ter119
þ
CD11b
þ
erythroid-myeloid cell aggregates were
enriched in F4/80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
compared with total BM macrophages (compare Fig. 2C
with Fig. 2A). Importantly, events representing single
cells according to low FSC-W contained very few
events that were double positive for Ter119 and CD11b
(Supplementary Figure E2, online only, available at www.
exphem.org) compared with cell aggregates (Fig. 2C),
showing that the BM cell aggregates preferentially contain
erythroid and myeloid cells. A 4-day treatment with G-CSF
in vivo caused a sixfold reduction in the number of in F4/
80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
macrophages
engaged in these erythroid-meyloid cell aggregates
(Fig. 2C) from 21,300 611,300 to 3,700 61,200 per fe-
mur (p50.02) in saline-treated and G-CSF-treated mice,
respectively. In G-CSF-mobilized BM, F4/80
Ly-6G
þ
granulocytes were the main myeloid cell type engaged in
Ter119
þ
CD11b
þ
cell aggregates.
Immunohistochemistry staining of consecutive sections
on mouse BM in steady-state clearly showed clusters of
Ter119
þ
erythroblasts associated with a very reticulated
ER-HR3
þ
macrophage at the center (Fig. 2D). This was
confirmed on dual staining of BM sections for Ter119
and ER-HR3 antigens, clearly showing long reticulated
ER-EHR3
þ
macrophages at the center of clusters of
Ter119
þ
nucleated erythroblasts (Supplementary
Figure E3, online only, available at www.exphem.org).
This is in situ confirmation that ER-HR3 antigen is ex-
pressed by erythroid island macrophage in the mouse BM.
To determine whether the effect of G-CSF on macro-
phages was directly mediated via the G-CSF receptor, we
sorted the following from the BM of untreated mice: F4/
80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
macrophages, F4/
80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
macrophages, and
F4/80
Ly-6G
þ
granulocytes as well as surface immunoglob-
ulin M (IgM)
þ
B220
þ
and surface IgM
B220
þ
Bcellsas
negative controls. We then extracted RNA. Quantitative
real-time RT-PCR showed that G-CSF receptor mRNA
(Csf3r) was abundantly expressed by these macrophages,
whereas it was absent in IgM
þ
and IgM
B cells (Fig. 2E).
This result suggests that G-CSF may act directly on erythroid
island macrophages as they express its cognate receptor.
Granulocyte colony-stimulating factor treatment does
not arrest splenic erythropoiesis
In contrast to the BM, G-CSF treatment significantly
increased proerythroblasts, all populations of erythroblasts,
and reticulocytes in the spleen (Fig. 3A and B), thereby ex-
plaining why the blood anemia is mild (Fig. 1D) despite a
profound blockage of medullar erythropoiesis. Spleens
were dissociated in culture medium with Ca
2þ
and Mg
2þ
using a GentleMACS Dissociator to preserve splenic
macrophage integrity. Flow cytometry of cell aggregates
with large FSC-W values showed the presence of
Ter119
þ
CD11b
þ
erythroid islands (albeit at a lower fre-
quency than in the BM) associated with F4/80
þ
VCAM-
1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
macrophages (Fig. 3C). In
sharp contrast to the BM, a 4-day treatment with G-CSF
in vivo increased three-point-fivefold the number of cell ag-
gregates between Ter119
þ
erythroid cells and CD11b
þ
myeloid cells per spleen (88,600 632,700 in saline vs.
307,700 6155,800 in G-CSF-treated mice, p50.03)
and did not significantly alter the number per spleen of
F4/80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
macrophages
engaged in these cell aggregates (Fig. 3C) (18,300
69,600 in saline vs. 21,300 66,100 in G-CSF-treated
Figure 2. (continued)(D) Double immunohistochemistry for ER-HR3 (brown) and Ter119 (red) antigens in femoral BM from mice treated with saline.
Reticulated ER-HR3
þ
macrophages are indicated by arrows at the center of Ter119
þ
(arrow heads) erythroid islands. (E) Quantification of Csf3r mRNA
by qRT-PCR on F4/80
Ly6-G
þ
granulocytes, F4/80
þ
VCAM1
þ
CD169
þ
ER-HR3
þ
Ly6-G
þ
macrophages, and F4/80
þ
VCAM1
þ
CD169
þ
ER-HR3
þ
Ly6-G
macrophages sorted from the BM of saline-treated mice. Sorted sIgM
and sIgM
þ
B cells were used as negative controls. Data are standardized to results
from F4/80
Ly6-G
þ
granulocytes and are mean 6SD of 4 mice (four independent sorts) per phenotype. Csf3r mRNA was undetectable in B cells.
*p#0.05; **p#0.01; ***p#0.001. G5Treated with G-CSF for 4 days; Sal 5treated with saline for 4 days.
552 R.N. Jacobsen et al./ Experimental Hematology 2014;42:547–561
mice, p50.62). By immunohistochemistry of serial sec-
tions, erythroid islands were detected exclusively in the
red pulp and, more specifically, in the subcortical region
of the spleen. Erythroid islands contained an ER-HR3
þ
macrophage at the center (Fig. 3D). Quantitative RT-PCR
on sorted splenic macrophages showed that F4/80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
macrophages also
expressed Csf3r mRNA (Fig. 3E).
Medullar erythropoiesis restarts after cessation of
granulocyte colony-stimulating factor
Since G-CSF is administered daily for 5–6 days in human
donors and has been shown to induce a slight (5%–10%)
but significant reduction in peripheral red blood cells
[31], we next measured the recovery of erythropoiesis
following cessation of G-CSF treatment. In mouse BM,
the number of erythroblasts did not recover before day
5–7 following cessation of a 4-day course of G-CSF admin-
istration (Fig. 4A). The excessive number of proerythro-
blasts sharply decreased between 24 hours and day 3
following G-CSF cessation and remained approximately
half of their normal number until day 7 after G-CSF
cessation.
With respect to the spleen, erythropoiesis remained
elevated, and normalization was more protracted than in
the BM, with a slow decrease in proerythroblasts and eryth-
roblast numbers during the 7 days following cessation of
G-CSF (Fig. 4C). This may explain why the blood
Figure 3. G-CSF treatment does not inhibit erythropoiesis in the spleen. (A) Representative flow cytometry dot-plots of mouse spleens after a 4-day treat-
ment with G-CSF or saline. The left panels show Ter119 versus CD71 amongst Ho
þ
nucleated cells (proerythroblasts were gated as Ter119
low
CD71
þ
, eryth-
roblasts as Ter119
þ
CD71
þ
, and polychromatic erythroblasts as Ter119
þ
CD71
low
). In the right panel, reticulocytes are gated as Ter119
þ
Ho
.(B)
Quantification of erythroid populations in spleens after a 4-day G-CSF or saline treatment. Data are mean 6SD of 4 mice per group from one representative
experiment among four independent repeats. Differences were evaluated with a ttest. (C) Representative phenotype of splenic cell aggregates (with high
forward scatter peak width) containing Ter119
þ
erythroid cells and CD11b
þ
myeloid cells in mice treated for 4 days with saline or G-CSF.
553R.N. Jacobsen et al. / Experimental Hematology 2014;42:547–561
concentration on hemoglobin decreased only slightly after a
4-day course of G-CSF (Fig. 4D).
With respect to BM macrophages, the number of F4/
80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
, which were
sharply suppressed after 4 days’ G-CSF treatment, recov-
ered by approximately 50% 24 hours after cessation of
G-CSF. However, complete recovery to steady-state levels
was delayed, as their number was still significantly
decreased 7 days after G-CSF cessation (Fig. 4B). This sug-
gests that a portion of BM erythroid island macrophages are
restored as early as 24 hours following G-CSF treatment,
which may kick-start differentiation of proerythroblasts
into erythroblasts and reticulocytes in the BM. However,
complete normalization requires more than 7 days after
cessation of G-CSF.
Macrophage depletion with clodronate-loaded
liposomes blocks erythropoiesis in both bone marrow
and spleen
To further test the hypothesis that F4/80
þ
VCAM-1
þ
ER-
HR3
þ
CD169
þ
Ly-6G
þ
macrophages depleted by G-CSF
treatment are erythroid island macrophages indispensable
to erythroblast maturation, we injected mice with
clodronate-loaded liposomes intravenously every other day
and sampled tissues after 4 days of this treatment. These lipo-
somes efficiently kill phagocytes in a variety of tissues,
including the BM and spleen [20]. Clodronate liposomes
reduced the number of F4/80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
macrophages twenty-eightfold, with almost complete
disappearance of F4/80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-
6G
þ
macrophages (Fig. 5C and D). Clodronate liposomes
did not indiscriminately ablate all F4/80
þ
macrophages in
the BM, as F4/80
þ
VCAM-1
macrophages remained rela-
tively insensitive to clodronate liposome treatment (Fig. 5C).
With respect to the erythroid linage, clodronate liposome
treatment also decreased the red coloration of BM flushes
(Fig. 1A), suggesting impaired medullar erythropoiesis.
Indeed, clodronate liposome administration reduced the
number of Ter119
þ
CD71
þ
erythroblasts four-point-
eightfold, Ter119
þ
CD71
low
polychromatic erythroblasts
nine-point-fourfold, Ter119
þ
CD71
Ho
þ
orthochromatic
erythroblasts two-point-fivefold and Ter119
þ
CD71
Ho
reticulocytes threefold (Fig. 5A and B). Similar to the result
for G-CSF treatment, Ter119
low
CD71
þ
Kit
Ho
þ
proery-
throblasts were increased three-point-sixfold by clodronate
liposomes (Fig. 5A and B). This suggests that, as with G-
CSF, clodronate liposome treatment blocks the maturation
of proerythroblasts into erythroblasts by depleting F4/
80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
macrophages at
the center of erythroid islands.
In sharp contrast to G-CSF treatment, clodronate lipo-
somes caused an extended depletion of Ter119
þ
CD71
þ
erythroblasts and Ter119
þ
CD71
low
polychromatic erythro-
blasts in the spleen, whereas reticulocyte numbers were un-
altered (Supplementary Figure E4, online only, available at
www.exphem.org). Therefore, unlike G-CSF, nonselective
depletion of phagocytes by clodronate liposomes impairs
erythropoiesis in both BM and spleen.
Figure 3. (continued)(D) Immunohistochemistry of ER-HR3 and Ter119 on consecutive sections of spleens from mice treated with saline. Yellow arrow in
top panel shows reticulated ER-HR3
þ
macrophage at the center of Ter119
þ
erythroid islands, indicated by green arrows in bottom panel. Note that erythroid
islands were enriched in the cortical area in the red pulp. (E) Quantification of Csf3r mRNA by qRT-PCR on F4/80
Ly6-G
þ
granulocytes, F4/
80
þ
VCAM1
þ
CD169
þ
ER-HR3
þ
Ly6-G
þ
macrophages, and and F4/80
þ
VCAM1
þ
CD169
þ
ER-HR3
þ
Ly6-G
macrophages sorted from the spleens of
saline-treated mice. Sorted sIgM
and sIgM
þ
B cells were used as negative controls. Data are standardized to results from F4/80
Ly6-G
þ
granulocytes
and are mean 6SD of 4 mice (four independent sorts) per phenotype. Csf3r mRNA was undetectable in B cells. *p#0.05; **p#0.01; ***p#0.001.
G5Treated with G-CSF for 4 days; Sal 5treated with saline for 4 days.
554 R.N. Jacobsen et al./ Experimental Hematology 2014;42:547–561
Figure 4. Kinetics of erythropoietic recovery in the BM following cessation of G-CSF. Mice were treated for 4 days with G-CSF and the numbers of (A)
erythroid cells, and (B) macrophages were quantified in the femoral BM by flow cytometry at the indicated time points after cessation of G-CSF. (C) Kinetics
of erythroid cell numbers in the spleen after cessation of G-CSF treatment. (D) Kinetics of blood hemoglobin concentration after cessation of G-CSF treat-
ment. Day 0 represents the last day of G-CSF. S on the abscissa indicates the values obtained from control mice treated for 4 days with saline instead of G-
CSF. Data are mean 6SD of 4 mice per group. Differences were evaluated with a ttest. *p#0.05; **p#0.01; ***p#0.001.
555R.N. Jacobsen et al. / Experimental Hematology 2014;42:547–561
Selective depletion of CD169
þ
macrophages blocks
erythropoiesis in both bone marrow and spleen
Finally, to further document the role of CD169
þ
macrophages
in erythropoiesis, we used Siglec1
DTR/þ
mice with a simian
DTR knocked in the mouse Siglec1 gene that encodes the
CD169 antigen. Diphtheria toxin (DPT) binds poorly to
mouse cells; however, the simian DTR has a high affinity
for DPT. As such, low doses of DPT (10–25 mg/kg) selectively
kill CD169
þ
macrophages, which express simian DTR in Si-
glec1
DTR/þ
mice [19]. Because DPT may cause an immune
reaction by itself, we compared the effect of DPT versus sa-
line in wild-type (Siglec1
þ/þ
) and Siglec1
DTR/þ
mice.
Notably, a 4-day course of DPT caused a slight but
measurable downregulation of CD169 expression on
Figure 5. Macrophage depletion with clodronate-loaded liposomes stops medullary erythropoiesis. (A) Representative flow cytometry dot-plots of
mouse BM after a 3-day treatment with clodronate-loaded liposomes or saline. The left panels show Ter119 versus CD71 among Ho
þ
nucleated cells
(proerythroblasts were gated as Ter119
low
CD71
þ
, erythroblasts as Ter119
þ
CD71
þ
, and polychromatic erythroblasts as Ter119
þ
CD71
low
). In the right
panel, reticulocytes are gated as Ter119
þ
Ho
.(B) Quantification of erythroid populations in the femoral BM after a 3-day clodronate liposome or
saline treatment. (C) Representative flow cytometry dot-plots of mouse BM after a 3-day treatment with clodronate-loaded liposomes or PBS-
loaded liposomes. BM F4/80
þ
macrophages were gated from CD11b
þ
myeloid cells and further subgated from expression of VCAM-1, CD169,
ER-HR3, and Ly6-G antigens.
556 R.N. Jacobsen et al./ Experimental Hematology 2014;42:547–561
macrophages in wild-type mice, but not of any of the
other antigens investigated herein (Fig. 6A). Likewise,
Siglec1
DTR/þ
mice treated with saline had reduced CD169
expression compared with saline-treated wild-type mice,
owing the loss of one of the two alleles of the Siglec1 gene
(Fig. 6A and B).
A 4-day course of 10 mg/kg DPT markedly reduced the
frequency and number of F4/80
þ
macrophages expressing
VCAM-1 (Fig. 6A) in Siglec1
DTR/þ
mice, resulting in a
twenty-six-point-eightfold reduction in the number of
F4/80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
macrophages
compared with Siglec1
DTR/þ
mice treated with saline
(Fig. 6A and B).
Similarly to treatment with G-CSF and clodronate lipo-
somes in wild-type mice, DPT treatment in Siglec1
DTR/þ
mice reduced the red coloration of flushed BM, whereas
DPT had a mild effect on the red coloration of the BM
from Siglec1
þ/þ
wild-type C57BL/6 mice (Supplementary
Figure E5, online only, available at www.exphem.org). As
recently reported [21], after DPT treatment, the numbers
of Ter119
þ
CD71
bright
erythroblasts, Ter119
þ
CD71
þ
poly-
chromatic erythroblasts, and Ter119
þ
CD71
reticulocytes
in BM were significantly reduced in the BM, with accumu-
lation of proerythroblasts (Supplementary Figure E5, online
only, available at www.exphem.org). However, in sharp
contrast to the previous two models, depletion of CD169
þ
macrophages had only a marginal effect on the number of
BM reticulocytes.
Finally, similarly to nonselective phagocyte depletion
with clodronate liposomes, selective depletion of CD169
þ
macrophages impaired splenic erythropoiesis with signifi-
cant reduction in the numbers of erythroblasts and poly-
chromatic erythroblasts (Supplementary Figure E6, online
only, available at www.exphem.org).
Discussion
Among differentiated cells of the body, macrophages
are the most functionally plastic [32,33]. They can fulfill
a large number of functions in innate immunity (e.g.,
phagocytosis of pathogens and dead cells), regulation of
adaptive immunity [34]; and tissue development, mainte-
nance, and repair [35,36]. Macrophages also play an essen-
tial role in pathogenesis caused by excessive inflammatory
responses and in various stages of cancer development
[37,38], such as the initial smoldering inflammation leading
to malignant transformation, tumor-associated neoangio-
genesis [39], metastasis [40,41], immune suppression
[42], and response to treatment [43]. Although macrophage
populations and function have been extensively studied in
these solid tissues, to our knowledge, relatively little is
known of macrophage populations and function in the BM.
We and others have recently reported that specific sub-
sets of BM macrophages are critical to the maintenance
of osteoblasts and bone formation at the endosteum
[16,22] and of HSC niches in the mouse [16]. Indeed,
depletion of macrophages stops bone formation and induces
HSC mobilization into the blood [16]. A subset of BM mac-
rophages has also been known for a long time to participate
to erythropoiesis, as suggested by the location of a central
macrophage in erythroid islands in mouse BM, spleen,
and liver [2,3,28]. Since G-CSF triggers HSC mobilization
by impairing the function of HSC niche–supportive macro-
phages in the BM [16,17], we explored the effect of G-CSF
on erythropoiesis in the mouse. Granulocyte colony-
stimulating factor caused the loss of BM macrophages
expressing VCAM-1, CD169, and ER-HR3 antigen, all of
which have been shown to be important to erythropoiesis
in the BM, spleen, or liver [5,21,28,30]. Treatment with
G-CSF caused a concomitant blockage of medullar erythro-
poiesis, with accumulation of proerythroblasts and sharp
reduction in the numbers of maturing erythroblasts and re-
ticulocytes, suggesting a blockade in proerythroblast matu-
ration and defective erythroblast maturation. This effect
was unique to the BM, as splenic erythropoiesis was not
inhibited, but increased by G-CSF treatment. We observed
that (1) G-CSF treatment caused a thirty-fivefold reduction
in the number of CD11b
þ
F4/80
þ
VCAM-1
þ
ER-
HR3
þ
CD169
þ
Ly-6G
þ
BM macrophages, (2) this macro-
phage phenotype was specifically enriched in BM cell
aggregates containing both myeloid and erythroid cells,
Figure 5. (continued)(D) Quantification of macrophage populations in femoral BM after a 3-day clodronate-loaded liposomes or saline treatment. Data are
mean 6SD of 4 mice per group from one representative experiment out of two independent repeats. Differences were evaluated with a ttest. *p#0.05;
**p#0.01; ***p#0.001. Clo 5Treated with clodronate liposome for 3 days; Sal 5treated with saline for 3 days.
557R.N. Jacobsen et al. / Experimental Hematology 2014;42:547–561
Figure 6. Depletion of CD169
þ
macrophages induces loss of erythroid island macrophages in the BM. Representative flow cytometry dot-plots of mouse
BM after a 4-day treatment with saline or DPT in wild-type mice or Siglec1
DTR/þ
mice. (A) Typical flow cytometry dot-plots showing BM F4/80
þ
macro-
phages gated from CD11b
þ
myeloid cells and further subgated from expression of VCAM-1, CD169, ER-HR3, and Ly6-G antigens. (B) Quantification of
macrophage populations in femoral BM after a 4-day treatment with saline or DPT in wild-type mice or Siglec1
DTR/þ
mice. DTR indicates the presence (þ)
or absence () of the Siglec1
DTR
mutant allele. DPT indicates treatment with DPT (þ) or saline (). Data are mean 6SD of 3–5 mice per group from one
representative experiment out of two independent repeats. Differences were evaluated with a ttest. *p#0.05; **p#0.01; ***p#0.001.
558 R.N. Jacobsen et al./ Experimental Hematology 2014;42:547–561
and (3) erythroid island macrophages expressed ER-HR3
antigen on BM sections. On the basis of these obser-
vations, we propose that CD11b
þ
F4/80
þ
VCAM-1
þ
ER-
HR3
þ
CD169
þ
Ly-6G
þ
macrophages represent erythroid
island macrophages that are necessary to erythroblast matu-
ration into reticulocytes, and that G-CSF treatment depletes
these macrophages, causing a blockade in medullar erythro-
poiesis. This conclusion is further substantiated by our
observation that treatment of wild-type mice with
clodronate-loaded liposomes, or treatment of Siglec1
DTR/þ
mice with DPT, caused a similar loss of F4/80
þ
VCAM-
1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
macrophages and blockade
of erythropoiesis. Our results are consistent with previous
observations that ER-HR3
þ
macrophages are highly phago-
cytic in vivo [27,44] and should therefore be depleted by
clodronate-loaded liposomes.
Erythroid island macrophages have been proposed to
facilitate Fe
2þ
transport into erythroblasts and actively
participate to their enucleation during the final stages of
erythroblast maturation. Our results with G-CSF and
clodronate-loaded liposomes and in Siglec1
DTR
mice
clearly indicate that, in addition to these roles, erythroid is-
land macrophages are necessary to the maturation of proer-
ythroblasts to erythroblasts. Whether this is linked to the
inability of erythroblasts to survive in the absence of
macrophage-mediated iron import or to secrete supportive
cytokines remains to be determined.
Our results are also consistent with a recent report
showing that erythropoietic island macrophages isolated
from the mouse BM are CD169
þ
and that their depletion
results in a blockade of medullar erythropoiesis [21]. Our
data identify more precisely the phenotype of these
erythroblast-supportive macrophages as CD11b
þ
F4/
80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
. Notably, G-CSF
and clodronate liposome treatments significantly increased
the number of CD11b
þ
F4/80
Ly-6G
þ
granulocytes,
consistent with our previous observations [16,18]. There-
fore, the Ly-6G antigen is not exclusive to granulocytes,
since it is also expressed by a subset of macrophages that
support erythroblasts (demonstrated herein) and HSCs
[16] in the BM.
Whist others have studied these erythropoietic island
macrophages using only one or two antigens, we have
examined the expression of a number of antigens at once,
allowing for a more precise identification of the erythropoi-
etic island macrophages. This will enable further studies on
these macrophages in steady-state as well as in disease
states. Anemia is one of the first symptoms associated
with a number of hematologic malignancies, including
chronic and acute myeloid leukemia, as well was myelo-
dysplastic syndrome. The possibility that erythropoietic is-
land macrophages may be involved or perturbed in the
initial stages of these diseases has not, to our knowledge,
been investigated because of the lack of precise markers
for these macrophages.
In respect to the function of these macrophage antigens
in regulating erythropoiesis, we noticed that Siglec1
DTR/DTR
mice, which are functionally knocked-out for the Siglec1
gene and CD169 expression, have normal numbers of pro-
erythroblasts, erythroblasts, and reticulocytes in their BM.
Therefore, the CD169 antigen, although useful to identify
or target erythroblast-supportive macrophages, is function-
ally dispensable for erythroblast maturation. We confirmed
that the ER-HR3 antigen, which identifies erythroid islands
in spleen and liver [5,27,28], also identifies erythroid island
macrophages in the BM. However, the exact identity of this
antigen and whether its expression is necessary to support
erythropoiesis remain unknown [44]. On the other hand,
it is well established that, within the BM hematopoietic
compartment, VCAM-1 is mainly expressed by macro-
phages [30] and mediates adhesion of erythroblasts to
erythroid island macrophages via integrin a4b1 expressed
by erythroblasts [45,46]. Furthermore conditional deletion
of the Vcam1 or Itga4 gene in mice impairs erythropoietic
recovery following cytotoxic or phenylhydrazine challenge
[29,30], whereas administration of function-blocking anti-
VCAM-1 antibody impairs erythropoietic recovery
following BM transplantation [21].
Although the BM is the main site of erythropoiesis in
adults in steady-state, the spleen and liver can become
important extramedullary erythropoietic organs when the
BM is compromised, as observed in some hematologic neo-
plasms [28,47]. Mobilizing treatment with G-CSF while
blocking medullar erythropoiesis significantly increased
splenic erythropoiesis. In contrast, both nonselective deple-
tion of phagocytes with clodronate liposomes and selective
ablation of CD169
þ
macrophages in the Siglec1
DTR/þ
models blocked splenic as well as medullar erythropoiesis,
probably because, in both these models, macrophages are
depleted regardless of their anatomical location. Interest-
ingly, however, Chow et al. have recently suggested that,
despite blockade of both medullar and splenic erythropoi-
esis in the Siglec1
DTR/þ
model, mice did not become
anemic as a result of a lengthening of erythrocyte turnover
following CD169
þ
macrophage depletion in the spleen
[21]. Such a mechanism is unlikely to be at play in G-
CSF-mobilized mice because G-CSF does not deplete
splenic macrophages.
It remains unclear how G-CSF causes the loss of
CD11b
þ
F4/80
þ
VCAM-1
þ
ER-HR3
þ
CD169
þ
Ly-6G
þ
macrophages in the BM without altering their number in
the spleen, since these cells express the receptor Csf3r
mRNA in both tissues. In the absence of monoclonal anti-
body specific for mouse G-CSF receptor, we could not
confirm these results at the protein level. It therefore re-
mains possible that the expression of the receptor at the
cell surface or signaling upon G-CSF binding is context
dependent and different in each tissue.
With respect to the relevance of our results to humans,
the effect of G-CSF on human erythropoiesis has not
559R.N. Jacobsen et al. / Experimental Hematology 2014;42:547–561
been extensively studied. However a similar effect may
occur; Juarez et al. recently reported that the number of
red blood cells also decreased following G-CSF-induced
mobilization by approximately 5%–10% in both in alloge-
neic (from 4.9 60.4 to 4.7 60.5 10
9
/mL, p510
4
) and
autologous (from 3.9 60.6 to 3.4 60.4 10
9
/mL,
p50.002) settings [31], despite the fact that human red
blood cells have a four times longer half-life than mouse
red blood cells (50–55 days [48] vs. 14 days) [49].
Although this remains to be confirmed experimentally on
BM samples from mobilized patients, G-CSF may also
cause a transient arrest in medullar erythropoiesis in hu-
mans by interfering with BM macrophages that support
erythropoiesis, with possible compensation in the spleen.
In conclusion, G-CSF doses used to mobilize HSCs in
transplantation donors transiently impair the function of
different subsets of tissue-supportive macrophages in the
mouse BM, particularly osteomacs that support bone-
forming osteoblasts [22,50], HSC niche-supportive macro-
phages [15–17] and erythropoietic island macrophages
(demonstrated herein). This further demonstrates that mac-
rophages are potent relays of innate immunity and inflam-
mation on osteogenic, hematopoietic, and erythropoietic
processes in the BM, and that treatments or agents that
affect these macrophages are likely to affect these three
processes concomitantly [12].
Acknowledgments
We thank Dr. Andrew Perkins for helpful discussions and Dalia
Khalil for cell sorting.
This work was supported by Project Grant No. 1046590 from
the National Health and Medical Research Council of Australia
and by the Mater Foundation. RN Jacobsen received an Australian
Post-graduate Award from the Commonwealth of Australia. JP
Levesque and IG Winkler received a Senior Research Fellowship
(No. 1044091) and a Career Development Fellowship (No.
1033736), respectively, from the National Health and Medical
Research Council.
Conflict of interest disclosure
No financial interest/relationships with financial interest relating
to the topic of this article have been declared.
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561R.N. Jacobsen et al. / Experimental Hematology 2014;42:547–561
Supplementary Figure E1. G-CSF-mediated depletion of medullar erythropoiesis as measured with CD44, CD45, and Ter119 antibodies. (A) Represen-
tative flow cytometry dot-plots of mouse BM after a 4-day treatment with G-CSF or saline. The left dot-plots show Ter119 vs. CD44 among gated
CD45
cells. Proerythroblasts were gated as Ter119
low
CD44
bright
(population I). Within the CD45
Ter119
þ
population, basophilic erythroblasts (population
II), polychromatic erythroblasts (population III), orthochromatic erythroblasts (population IV), reticulocytes (population V), and erythrocytes (population VI)
were gated according to decreasing forward scatter and CD44 expression. (B) Quantification of erythroid populations in the femoral BM after a 4-day course
of saline (Sal) or G-CSF (G) treatment. n54 mice per group. Differences were evaluated with a ttest. *p#0.05; **p#0.01; ***p#0.001.
Supplementary Figure E2. Cell aggregates in the BM are enriched in clusters made of Ter119
þ
erythroid cells and CD11b
þ
myeloid cells. Representative
phenotype of BM cell aggregates with high forward scatter peak width (in the top panels) and BM cell singlets with lower forward scatter peak width (in the
lower panels) containing Ter119
þ
erythroid cells and CD11b
þ
myeloid cells. While 21% of cell aggregates in BM from saline-treated mice contain clusters of
Ter119
þ
erythroid cells and CD11b
þ
cells, only 0.31% of cells in the singlet gate contained Ter119
þ
CD11b
þ
events.
561.e1 R.N. Jacobsen et al./ Experimental Hematology 2014;42:547–561
Supplementary Figure E3. ER-HR3
þ
macrophages at the Centre of
erythroid islands. Double immunohistochemistry for ER-HR3 (brown)
and Ter119 (red) antigens in femoral BM from mice treated with saline.
Reticulated ER-HR3
þ
macrophages are indicated by arrows at the center
of clusters of Ter119
þ
erythroblasts (arrowheads).
Supplementary Figure E4. Macrophage depletion with clodronate-loaded liposomes stops splenic erythropoiesis. (A) Representative flow cytometry dot-
plots of spleens after a 3-day treatment with clodronate-loaded liposomes or saline. The left panels show Ter119 vs. CD71 amongst Ho
þ
nucleated cells.
(Proerythroblasts were gated as Ter119
low
CD71
þ
, erythroblasts as Ter119
þ
CD71
þ
, polychromatic erythroblasts as Ter119
þ
CD71
low
). In the right panel,
reticulocytes are gated as Ter119
þ
Ho
.(B) Quantification of erythroid populations in the spleen after a 3-day clodronate liposome (Clo) or saline (Sal)
treatment. Data are mean 6SD of 4 mice per group from one representative experiment out of two independent repeats. Differences were evaluated
with a ttest. *p#0.05; **p#0.01; ***p#0.001.
561.e2R.N. Jacobsen et al. / Experimental Hematology 2014;42:547–561
Supplementary Figure E5. Depletion of CD169
þ
macrophages stops medullary erythropoiesis. (A) Representative flow cytometry dot-plots of mouse BM
after a 4-day treatment with saline or DPT in wild-type mice or Siglec1
DTR/þ
mice. The left panels show Ter119 vs. CD71 amongst Ho
þ
nucleated cells.
(Proerythroblasts were gated as Ter119
low
CD71
þ
, erythroblasts as Ter119
þ
CD71
þ
, polychromatic erythroblasts as Ter119
þ
CD71
low
). In the right panel
reticulocytes are gated as Ter119
þ
Ho
.(B) Quantification of erythroid populations in the femoral BM after a 4-day treatment with saline or DPT in
wild-type mice or Siglec1
DTR/þ
mice. DTR indicates the presence (þ) or absence () of the Siglec1
DTR
mutant allele. DPT indicates treatment with
DPT (þ) or saline (). Data are mean 6SD of 4 mice per group from one representative experiment out of two independent repeats. Differences were
evaluated with a ttest. (C) Photograph of mouse femoral BM flushed into 1 mL PBS after 4-day treatment with saline or DPT in wild-type and Si-
glec1
DTR/þ
mice. Note the loss of red coloration following DPT treatment in Siglec1
DTR/þ
mice. *p#0.05; **p#0.01; ***p#0.001.
561.e3 R.N. Jacobsen et al./ Experimental Hematology 2014;42:547–561
Supplementary Figure E6. Depletion of CD169þmacrophages stops splenic erythropoiesis. (A) Representative flow cytometry dot-plots of spleens after a
4-day treatment with saline or DPT in wild-type mice or Siglec1
DTR/þ
mice. The left panels show Ter119 vs. CD71 amongst Ho
þ
nucleated cells. (Proer-
ythroblasts were gated as Ter119
low
CD71
þ
, erythroblasts as Ter119
þ
CD71
þ
, polychromatic erythroblasts as Ter119
þ
CD71
low
). In the right panel reticulo-
cytes are gated as Ter119
þ
Ho
.(B) Quantification of erythroid populations in the spleen after a 4-day treatment with saline or DPT in wild-type mice or
Siglec1
DTR/þ
mice. DTR indicates the presence (þ) or absence () of the Siglec1
DTR
mutant allele. DPT indicates treatment with DPT (þ) or saline (). Data
are mean 6SD of 4 mice per group from one representative experiment out of two independent repeats. Differences were evaluated with a ttest. *p#0.05;
**p#0.01; ***p#0.001.
561.e4R.N. Jacobsen et al. / Experimental Hematology 2014;42:547–561
... F4/80, VCAM1, CD169, and CD11b have been reported as markers for EBI Mφs, based on non-imaging flow cytometry studies of events double positive for CD71 and F4/80. 12,13 We utilized gravity Downloaded from http://ashpublications.org/blood/article-pdf/doi/10.1182/blood.2022015724/1910148/blood.2022015724.pdf by CINCINNATI CHILDRENS HOSPITAL MEDICAL CENTER, Theodosia Kalfa on 18 September 2022 sedimentation to isolate BM clusters enriched for EBIs and performed unbiased analysis by Imaging Flow Cytometry (IFC), focused on the morphological identity of a macrophage tightly associated with surrounding erythroblasts. ...
... 30 This result is consistent with substantial heterogeneity within the F4/80+ EBI macrophage population, 14 and also points to the difficulty isolating EBI macrophages from other cells for studies based solely on immunophenotype. 13,33 The EBI balances erythropoiesis and granulopoiesis Based on our observation of granulocytic maturation within EBIs, we hypothesized that this common niche might regulate the balance between these two lineages. To test this hypothesis, we used biological models of altered granulopoiesis to evaluate concurrent changes in erythropoiesis within EBIs/EMBIs using IFC. ...
... The was run on GEX-only profiles following removal of ambient background associated RNAs with SoupX (contamination fraction 15%). 13 ICGS was performed using the software default options and Euclidean clustering, with no forced resolution parameter, in addition to a PageRank downsampling threshold of 5,000 cells. Cell population names were revised from the original ICGS2 ...
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The erythroblastic island (EBI), composed of a central macrophage surrounded by maturing erythroblasts, is the erythroid precursor niche. Despite numerous studies, its precise composition is still unclear. Using multispectral imaging flow cytometry (IFC), in vitro island reconstitution, and single cell RNA-sequencing (scRNA-seq) of adult mouse bone marrow EBI-component cells enriched by gradient sedimentation, we present evidence that the CD11b+-cells present in the EBIs are neutrophil precursors specifically associated with bone marrow EBI macrophages, indicating that erythro-(myelo)-blastic islands are a site for both terminal granulopoiesis and erythropoiesis. We further demonstrate that the balance between these dominant and terminal differentiation programs is dynamically regulated within this bone marrow niche by pathophysiological states, favoring granulopoiesis during anemia of inflammation, or erythropoiesis after erythropoietin (Epo) stimulation. Finally, by molecular profiling, we reveal the heterogeneity of EBI macrophages by Cellular Indexing of Transcriptome and Epitopes (CITE)-sequencing of mouse bone marrow EBIs at baseline and after Epo-stimulation in vivo and provide a searchable, online viewer of this data characterizing the macrophage subsets serving as hematopoietic niches. Taken together, our findings demonstrate that EBIs serve a dual role as niches for terminal erythropoiesis and granulopoiesis and the central macrophages adapt to optimize production of red blood cells or neutrophils.
... Many hematopoietic growth factors regulate erythropoiesis by affecting the function of EBI macrophages. Erythropoietin (EPO) acts on both erythroid cells and EBI macrophages simultaneously to ensure efficient erythropoiesis [17,19]; Granulocyte colony-stimulating factor (G-CSF) blocks medullary erythropoiesis by depleting EBI macrophages in mouse BM [20,21]. These functional studies strongly suggest that hematopoietic growth factors can regulate erythropoiesis by affecting the roles of EBI macrophages. ...
... Additionally, studies have also shown that the effect of G-CSF on HSC niches is mediated in part by a subpopulation of BM macrophages [26]. G-CSF also causes a significant loss of BM macrophages expressing VCAM-1, CD169, and ER-HR3 and blocks medullary erythropoiesis in BM [20]. Furthermore, using imaging flowcytometry (IFC), Joshua Tay et al. found that G-CSF reduces EBI frequency in the BM by more than 100 times [21]. ...
... In the present study, we tested the roles of GM-CSF in human and mouse EBI formation, finding that GM-CSF significantly decreases EBI formation both in vitro and in vivo. G-CSF also impairs EBI formation in vivo [20,21]. Conversely, previous studies have shown that G-CSF induces splenic erythropoiesis at the same time [20]. ...
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Anemia is a significant complication of chronic inflammation and may be related to dysregulated activities among erythroblastic island (EBI) macrophages. GM-CSF was reported to be upregulated and attracted as a therapeutic target in many inflammatory diseases. Among EBIs, we found that the GM-CSF receptor is preferentially and highly expressed among EBI macrophages but not among erythroblasts. GM-CSF treatment significantly decreases human EBI formation in vitro by decreasing the adhesion molecule expression of CD163. RNA-sequence analysis suggests that GM-CSF treatment impairs the supporting function of human EBI macrophages during erythropoiesis. GM-CSF treatment also polarizes human EBI macrophages from M2-like type to M1-like type. In addition, GM-CSF decreases mouse bone marrow (BM) erythroblasts as well as EBI macrophages, leading to a reduction in EBI numbers. In defining the molecular mechanism at work, we found that GM-CSF treatment significantly decreases the adhesion molecule expression of CD163 and Vcam1 in vivo. Importantly, GM-CSF treatment also decreases the phagocytosis rate of EBI macrophages in mouse BM as well as decreases the expression of the engulfment-related molecules Mertk, Axl, and Timd4. In addition, GM-CSF treatment polarizes mouse BM EBI macrophages from M2-like type to M1-like type. Thus, we document that GM-CSF impairs EBI formation in mice and humans. Our findings support that targeting GM-CSF or reprogramming EBI macrophages might be a novel strategy to treat anemia resulting from inflammatory diseases.
... BM F4/80 + Ly6G + events exemplify the macrophage origin of F4/80 + remnants F4/80 + Ly6G + events in BM detected by traditional flow cytometry have previously been identified and purified as erythroblastic island macrophages (EIMs), also expressing VCAM-1, CD169, and ER-HR3 (Jacobsen et al., 2014;Li et al., 2019). Herein, imaging flow cytometry indicated that this dual expression is a consequence of macrophage-remnant binding to Ly6G + neutrophils ( Figures 3A and 3B) and therefore is not an EIM-distinguishing phenotype. ...
... Key observations made herein were that the cell-attached macrophage remnants contained not only membrane and associated proteins at high molecular density but also intracellular contents, including significant amounts of mRNA. This technical phenomenon has contributed to the misassignment of macrophage identity to flow-cytometry-gated populations in hematopoietic tissues, including our own studies reporting F4/80 + Ly6G + cell events as EIMs (Jacobsen et al., 2014;Kaur et al., 2018). Indeed, Li et al. (2019) used a distinct approach to isolate and profile F4/ 80 + EIMs based upon an Epor-EGFP transgene, but our analysis herein of their data revealed strong enrichment for immature myeloid transcripts in the putative EIMs. ...
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Mouse hematopoietic tissues contain abundant tissue-resident macrophages that support immunity, hematopoiesis, and bone homeostasis. A systematic strategy to characterize macrophage subsets in mouse bone marrow (BM), spleen, and lymph node unexpectedly reveals that macrophage surface marker staining emanates from membrane-bound subcellular remnants associated with unrelated cells. Intact macrophages are not present within these cell preparations. The macrophage remnant binding profile reflects interactions between macrophages and other cell types in vivo. Depletion of CD169⁺ macrophages in vivo eliminates F4/80⁺ remnant attachment. Remnant-restricted macrophage-specific membrane markers, cytoplasmic fluorescent reporters, and mRNA are all detected in non-macrophage cells including isolated stem and progenitor cells. Analysis of RNA sequencing (RNA-seq) data, including publicly available datasets, indicates that macrophage fragmentation is a general phenomenon that confounds bulk and single-cell analysis of disaggregated hematopoietic tissues. Hematopoietic tissue macrophage fragmentation undermines the accuracy of macrophage ex vivo molecular profiling and creates opportunity for misattribution of macrophage-expressed genes to non-macrophage cells.
... On the one hand, fusion between cells of the monocyte/macrophage lineage leads to the formation of osteoclasts, which are the only cells with the ability to dissolve bone tissue (26). On the other hand, ablation of macrophages leads to loss of endosteal osteoblasts, reduction in the number of bone marrow mesenchymal stem cells (BMSCs), decrease in the ability of BMSCs to differentiate into osteoblasts, and attenuation of parathyroid hormone-induced trabecular bone anabolism (27)(28)(29)(30). The macrophage-osteoclast axis plays an essential role in osteoimmunity, regulating the coupling of bone resorption and bone formation (31). ...
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Postmenopausal osteoporosis (PMOP) is characterized by the uncoupling of bone resorption and bone formation induced by estrogen deficiency, which is a complex outcome related to estrogen and the immune system. The interaction between bone and immune cells is regarded as the context of PMOP. Macrophages act differently on bone cells, depending on their polarization profile and secreted paracrine factors, which may have implications for the development of PMOP. PMOP, rheumatoid arthritis (RA), and Alzheimer’s disease (AD) might have pathophysiological links, and the similarity of their pathological mechanisms is partially visible in altered macrophages and cytokines in the immune system. This review focuses on exploring the pathological mechanisms of PMOP, RA, and AD through the roles of altered macrophages and cytokines secretion. First, the multiple effects on cytokines secretion by bone-bone marrow (BM) macrophages in the pathological mechanism of PMOP are reviewed. Then, based on the thought of “different tissue-same cell type-common pathological molecules-disease pathological links-drug targets” and the methodologies of “molecular network” in bioinformatics, highlight that multiple cytokines overlap in the pathological molecules associated with PMOP vs. RA and PMOP vs. AD, and propose that these overlaps may lead to a pathological synergy in PMOP, RA, and AD. It provides a novel strategy for understanding the pathogenesis of PMOP and potential drug targets for the treatment of PMOP.
... In vivo macrophage depletion promotes increased mobilization of HSC (11). Moreover, Hur et al., revealed that CD234 + macrophages can interact with CD82 + long-term HSC to support quiescence in the endosteal region (12), while others identified erythroblastic island macrophages to promote erythropoiesis (13,14). Thus, the BM harbors diverse macrophage populations, which can be exploited by LSCs. ...
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While it is increasingly becoming clear that cancers are a symbiosis of diverse cell types and tumor clones, the tumor microenvironment (TME) in acute myeloid leukemias (AML) remains poorly understood. Here, we uncover the functional and prognostic relevance of an M2-polarized macrophage compartment. Intra bone marrow co-injection of M2d-macrophages together with leukemic blasts that fail to engraft on their own now induce fatal leukemia in mice. Even a short-term two-day in vitro exposure to M2d macrophages can 'train' leukemic blasts after which cells are protected against phagocytosis, display increased mitochondrial metabolism and improved in vivo homing, resulting in full-blown leukemia. We developed an M2d-based biomarker panel that outperforms currently used AML prognosis predictors. Our study provides insight into the mechanisms by which the immune landscape contributes to aggressive leukemia development and provides alternatives for effective targeting strategies.
... Les macrophages résidents de la moelle osseuse sont également connus pour leur rôle dans la régulation de l'érythropoïèse ( Figure 19). La déplétion de ces macrophages par le G-CSF ou le Flt3L, ou encore dans les modèles clodronateliposomes ou CD169-DTR, entraine un blocage de l'érythropoïèse (Jacobsen et al., 2014(Jacobsen et al., , 2016Tay et al., 2020) et une diminution du nombre d'érythroblastes dans la moelle osseuse et dans la rate . ...
Thesis
Avec plus d’un patient sur deux qui en bénéficie, la radiothérapie est l’une des méthodes les plus utilisées dans le traitement des cancers. Malgré son efficacité dans l’éradication des tumeurs, le principal inconvénient de cette technique est la toxicité qu’elle peut entrainer sur les tissus sains environnants. Au niveau de la moelle osseuse, qui contient les cellules souches hématopoïétiques (CSH), cette toxicité peut être très délétère. En effet, les CSH sont responsables de l’hématopoïèse tout au long de la vie d’un individu, d’où l’importance de les préserver. Ces cellules sont localisées dans un microenvironnement cellulaire, appelé niche hématopoïétique, jouant un rôle majeur dans leur protection et le maintien de leur intégrité. Au sein de cette niche, les macrophages résidents de la moelle osseuse, caractérisés par l’expression du marqueur de surface CD169, ont montré un rôle à la fois dans le maintien des CSH dans leur niche, mais aussi dans la protection des cellules vis-à-vis d’un stress oxydatif. Dans ce contexte, mon projet de thèse avait pour but de définir le rôle de ces macrophages résidents de la moelle osseuse dans la réponse des CSH à une irradiation corps entier (TBI) de 2 Gy, dose couramment utilisée en radiothérapie fractionnée. Afin de répondre à cette question, j’ai utilisé deux modèles de souris déplétées en macrophages CD169+ (Mϕ CD169+) : un modèle pharmacologique (clodronate-liposomes) et un modèle génétique (souris CD169DTR/+). Dans ces deux modèles, j’ai montré qu’une déplétion des Mϕ CD169+ avant l’irradiation entraine à court-terme une diminution moins importante du réservoir de CSH, accompagnée d’une diminution de leur apoptose et d’une absence d’espèces réactives de l’oxygène (ROS) généralement induites par l’irradiation. A plus long-terme, l’absence de Mϕ CD169+ permet un rétablissement complet d’un réservoir fonctionnel de CSH après irradiation. L’ensemble de ces résultats démontre que la présence des macrophages résidents lors d’une irradiation a un rôle délétère en diminuant la réserve de CSH au sein de la moelle osseuse. Afin de mettre en évidence le ou les mécanismes provoquant cet effet délétère, je me suis intéressée à la réponse directe des Mϕ CD169+ à l’irradiation, en ciblant particulièrement les phénomènes liés au stress oxydatif. J’ai observé que la TBI entraine une augmentation de la proportion de Mϕ CD169+ qui produisent de l’oxyde nitrique (NO), une des caractéristiques de la réponse de type pro-inflammatoire des Mϕ. Cette augmentation est corrélée à une augmentation des CSH ayant des peroxynitrites, oxydants extrêmement cytotoxiques issus de la réaction entre les NO et les ROS. L’utilisation de modulateurs négatifs ou positifs des NO a montré qu’après une TBI de 2 Gy, la diminution de la production de NO par les Mϕ CD169+ permet de limiter l’apoptose des CSH et de restaurer leur nombre, alors que l’augmentation de NO dans l’environnement médullaire entraîne une diminution de leur nombre. Cette étude démontre que l’irradiation accroit la production de NO par les macrophages résidents de la moelle, entrainant un effet nocif sur la réserve de CSH. Ces nouvelles données identifient le macrophage résident comme un candidat potentiel pour moduler les effets de l’irradiation sur les CSH.
... For example, the Kalfa lab analyzed single cells derived from isolated whole erythroblastic islands (Seu et al., 2018). Their data support the idea that non-F4/80 cells (ie, F4/80-Ly6G+ granulocytes) may play a role in a separate subset of island function, particularly as related to myelopoiesis following anemia of inflammation, leading to a dynamic antagonistic balance between granulopoiesis and erythropoiesis, a notion supported by the dramatic effects of G-CSF treatment on suppression of erythropoiesis (Jacobsen et al., 2014;Jacobsen et al., 2015). These data are consistent with imaging multi-color fluorescence analyses showing that F4/80, VCAM1, and CD169 (Siglec1), but not CD11b (Itgam) or Ly6G, are expressed in erythroblastic islands, while at the same time, the CD11b+Ly6G+F4/80-cells can be found "at the periphery" of a subset of these aggregates (Tay et al., 2020). ...
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... Finally, it has recently been reported in zebrafish that VCAM-1 + patrolling macrophages can interact with HSCs in an a 4 b 1 dependent manner and contribute to their retention in the niche (35). This study confirms earlier findings in mouse models showing that macrophages contribute to HSC retention within niches through integrin-mediated interactions (36)(37)(38). ...
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